Chromosome Congression by Kinesin-5Motor-Mediated Disassemblyof Longer Kinetochore MicrotubulesMelissa K. Gardner,1 David C. Bouck,2 Leocadia V. Paliulis,2 Janet B. Meehl,3 Eileen T. O’Toole,3 Julian Haase,2

Adelheid Soubry,2 Ajit P. Joglekar,2 Mark Winey,3 Edward D. Salmon,2 Kerry Bloom,2 and David J. Odde1,*1Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA2Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA3MCD Biology, University of Colorado, Boulder, CO 80301, USA*Correspondence: oddex002@umn.edu

DOI 10.1016/j.cell.2008.09.046

SUMMARY

During mitosis, sister chromatids congress to thespindle equator and are subsequently segregatedvia attachment to dynamic kinetochore microtubule(kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chro-mosome congression. Here we find in budding yeastthat the widely conserved kinesin-5 sliding motorproteins, Cin8p and Kip1p, mediate chromosomecongression by suppressing kMT plus-end assemblyof longer kMTs. Of the two, Cin8p is the major effec-tor and its activity requires a functional motor do-main. In contrast, the depolymerizing kinesin-8 mo-tor Kip3p plays a minor role in spatial regulation ofyeast kMT assembly. Our analysis identified a modelwhere kinesin-5 motors bind to kMTs, move to kMTplus ends, and upon arrival at a growing plus endpromote net kMT plus-end disassembly. In conclu-sion, we find that length-dependent control of netkMT assembly by kinesin-5 motors yields a simpleand stable self-organizing mechanism for chromo-some congression.

INTRODUCTION

A central question in biology is how replicated chromosomes are

properly segregated during mitosis, such that exactly one copy

of each chromosome moves to each of the two daughter cells

(Nicklas, 1997). In eukaryotes, chromosome-associated kineto-

chores attach to dynamic microtubule (MT) plus ends, while

MT minus ends in turn generally attach to spindle poles (Inoue

and Salmon, 1995). Once properly bioriented, so that one kinet-

ochore is mechanically linked via one or more MTs to one pole

and its sister kinetochore linked to the opposite pole, the sister

chromosomes move toward the equator of the mitotic spindle,

a process known as congression (Figure 1A). In budding yeast,

congression of bioriented sister chromosomes requires that

the associated kinetochore microtubule (kMT) plus ends add tu-

894 Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc.

bulin subunits efficiently when they are near the poles (i.e., when

kMTs are short) but inefficiently when near the equator (i.e., when

kMTs are relatively long; Gardner et al., 2005; Pearson et al.,

2006; Sprague et al., 2003; Figure 1A). The origin of this spatial

gradient in net kMT plus-end assembly is currently unknown.

One possible explanation for the spatial gradient of kMT net

assembly derives from studies with the budding yeast kinesin-8

molecular motor Kip3p, which showed that processive plus-

end-directed depolymerizing motors could result in length-

dependent disassembly of MTs in vitro (Howard and Hyman,

2007; Varga et al., 2006). However, to date there has not been

an in vivo observation of length-dependent MT regulation via

Kip3p or any other motor. Recent in vivo studies of Kip3p

showed that cytoplasmic microtubule dynamics are altered by

Kip3p, and that KIP3 deletion results in spindle positioning de-

fects (Gupta et al., 2006). Whether these in vivo effects resulted

from the length-dependent control of MT assembly as proposed

by Varga et al. (2006) was not clear. As a result, it is not clear at

this point whether this interesting phenomenon identified in vitro

by Varga et al. plays an important role in a biological process.

We now find that the kinesin-5 molecular motors, mainly Cin8p

and, to a lesser extent, Kip1p, exhibit length-dependent control

of MT assembly and so mediate the spatial gradient in kMT plus-

end net assembly that drives congression during mitosis. Since

their discovery, kinesin-5 motors have been viewed as mitosis-

specific sliding motors that crosslink antiparallel MTs and exert

outward extensional forces on the poles (Hoyt et al., 1992; Kapi-

tein et al., 2005; Roof et al., 1992; Saunders and Hoyt, 1992; Tao

et al., 2006). The results were surprising to us because kinesin-5

(Eg5 in vertebrates) is not known to affect microtubule assembly.

Surprisingly, the disassembly-promoting activity that we now re-

port is not specific to kMTs since we found that Cin8p promotes

disassembly of cytoplasmic astral MTs (aMTs) as well.

Because the kinesin-5-mediated length-dependent kMT dis-

assembly is similar to that described by Varga et al. for Kip3p

on MTs in vitro, we examined the role of Kip3p in controlling

kMT assembly in vivo. We found that Kip3p only weakly affects

kMT dynamics and is not a major mediator of congression. Our

computer simulations show that the difference in kinesin-5 and

kinesin-8 behavior can be accounted for simply by the bipolar

ability of tetrameric kinesin-5 to crosslink microtubules, a

Figure 1. Cin8p Organizes Metaphase Yeast Kinetochores in a Manner Consistent with Length-Dependent Suppression of Net kMT Plus-End

Assembly

(A) A spatial gradient in net kMT plus-end assembly mediates kinetochore congression in yeast. Kinetochores (cyan) congress to attractor zones (yellow arrows)

on either side of the spindle equator (dotted line) during yeast metaphase via the plus-end assembly dynamics of kMTs (black). The kMT plus-end assembly

dynamics are spatially regulated such that plus-end assembly is favored near the poles (gray) when kMTs are relatively short (favorable assembly zone shown

as green gradient) and suppressed near the spindle equator (dotted line) when kMTs are relatively long (assembly suppression zone shown as red gradient).

(B)A computational modelofyeast metaphasekMTplus-enddynamics predicts that deletionof the length-dependent promoterofkMT disassembly will result in longer

kMTs and disorganized kinetochores. Conversely, overexpression will result in shorter kMTs and focused kinetochore clusters. All model parameters as in Table S2.

(C)CIN8deletion results inkinetochore disorganizationand the net shiftingofkinetochores toward the spindleequator, suggesting thatkMTs are onaverage longer than

they are in WT cells (red, Spc29-CFP pole marker; green, Cse4-GFP kinetochore marker) (scale bar 1000 nm, error bars, SEM).

(D) Cin8p overexpression results in clusters of kinetochores near the SPBs, suggesting that kMTs are shorter than in WT cells (error bars, SEM).

Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc. 895

property lacking in dimeric kinesin-8. This naturally biases kine-

sin-5 toward kMT plus ends and kinesin-8 toward interpolar MT

plus ends. Together, the results presented here suggest a simple

explanation for metaphase chromosome congression in budding

yeast by identifying a microtubule length-dependent disassem-

bly-promoting activity in vivo that is associated with kinesin-5

molecular motors.

RESULTS

Simulated Phenotypes of a Disrupted Gradient in NetkMT Plus-End AssemblyMTs self-assemble from ab-tubulin heterodimers via an unusual

process called ‘‘dynamic instability’’ where MTs grow at

a roughly constant rate, then abruptly and stochastically switch

to shortening at a roughly constant rate, then switch back to

growth again, and so forth. The switch from growth to shortening

is called ‘‘catastrophe,’’ and the switch from shortening

to growth is called ‘‘rescue’’ (Desai and Mitchison, 1997). To-

gether, the four parameters of dynamic instability, growth rate

(Vg [ = ] mm/min), shortening rate (Vs [ = ] mm/min), catastrophe

frequency (kc [ = ] 1/min), and rescue frequency (kr [ = ] 1/min),

define the net assembly state of MTs. If the mean length added

during a growth phase (Lg = Vg/kc [ = ] mm) exceeds the mean

length lost during shortening (Ls = Vs/kr [ = ] mm), then there will

be net growth; otherwise there will be net shortening. If the pa-

rameters depend upon position in the cell, then there can exist

net growth in one part of the cell and net shortening in another

part of the cell. At the transition between these regions there

will be no net growth, and this will create an attractor for plus

ends, provided net growth is favored for short MTs and net short-

ening favored for long MTs (Figure 1A).

Our previous studies showed that net kMT plus-end assembly

in budding yeast metaphase spindles is favored for plus ends lo-

cated near the spindle pole bodies (SPBs) (i.e., for short kMTs)

and inhibited for plus ends near the equator (i.e., for longer

kMTs) (Figure 1A; Gardner et al., 2005; Pearson et al., 2006;

Pearson et al., 2004; Sprague et al., 2003). This spatial control

over net kMT assembly was most readily explained using a ca-

tastrophe gradient in kMT plus-end assembly that creates two

attractor points where there is no net assembly, one attractor

in each half-spindle (Figure 1B, left, dotted line denotes the loca-

tion of the attractor point for one half-spindle). These two attrac-

tors establish the bilobed distribution of kinetochores into the

two distinct clusters that are characteristic of the congressed

metaphase spindle (Figure 1A).

We were interested in identifying the molecules responsible for

this net assembly gradient, and so we simulated the expected

phenotypes for changes in expression level of a putative spatial

regulator of net kMT plus-end assembly. Figure 1B (left) shows

a simulation of a wild-type (WT) budding yeast metaphase spin-

dle where kMT plus-end assembly is favored near the SPB

(where kMTs are short) and suppressed near the spindle equator

(where kMTs are longer). Note that in the haploid budding yeast

there are 16 kMTs emanating from each SPB, for a total of

32 kMTs, and �8 longer interpolar MTs (iMTs), for a total of

40 MTs in the metaphase spindle (for the sake of clarity the

iMTs are omitted from the animation in Figure 1). If a molecule

896 Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc.

promoted disassembly of longer kMTs in WT cells and was

then deleted, the predicted phenotype would be longer kMTs

with kinetochores more broadly distributed along the spindle,

as depicted in Figure 1B, center. Conversely, overexpression

of a spatial assembly regulator would produce very short kMTs

with highly focused clusters of kinetochores near the SPBs, as

depicted in Figure 1B, right. These model predictions establish

specific requirements for experimental identification of a spatial

kMT plus-end assembly regulator.

The Yeast Kinesin-5 Motors, Cin8p and Kip1p, ControlKinetochore PositionsWhile studying various deletion mutants, we observed that cin8D

mutants lost the clustering of kinetochores within each half-

spindle (measured by labeling of a kinetochore component,

Cse4-GFP, in live cells), as shown in Figure 1C, consistent with

an earlier report (Tytell and Sorger, 2006). Quantitative analysis

of experimental Cse4-GFP fluorescence revealed that the peak

fluorescence intensity also shifted toward the spindle equator,

as predicted by simulations used to model deletion of a molecule

that promotes net kMT plus-end disassembly of longer kMTs

(Figure 1C, p = 0.82, where p is the probability that the experi-

mental Cse4-GFP fluorescence distribution curve is consistent

with the simulated curve; see Experimental Procedures for cal-

culation procedure). The simulated Cse4-GFP fluorescence dis-

tribution was obtained by convolution of the simulated fluoro-

phore positions with the imaging system point spread function

and noise, a computational process we call ‘‘model-convolu-

tion’’ (Gardner et al., 2007; Sprague et al., 2003). Deletion of

the other yeast kinesin-5 motor, KIP1, had a similar, but weaker,

phenotype to cin8D, with a moderate shift of kinetochores to-

ward the spindle equator (Figure S1A available online). We

note that the effects of CIN8 deletion were not due to the well-

known moderate decrease in steady-state spindle length (Hilde-

brandt and Hoyt, 2000; Hoyt et al., 1992; Saunders et al., 1997;

Saunders and Hoyt, 1992) since we selected spindle lengths that

were equal for both WT and cin8D cells (although results were

similar regardless of the spindle length population analyzed;

Figure S2). To further establish that the effects of CIN8 deletion

were not due to changes in metaphase spindle lengths, we per-

formed separate experiments using histone H3 repression mu-

tants, which make centromeric chromatin more compliant and

thus increase average spindle length (Bouck and Bloom, 2007).

In these longer spindles, WT kinetochores were still bilobed while

cin8D kinetochores were still disorganized (Figure S1B), showing

an insensitivity of the disorganization phenotype to spindle

length. In addition, bim1D mutant spindles with short spindle

lengths (similar to cin8D mutant spindle lengths, Figure S3) did

not result in spindle disorganization (Figure S3). We conclude

that kinetochores are declustered in kinesin-5 deletion mutants,

independent of the spindle length.

If Cin8p mediates net kMT plus-end disassembly, then Cin8p

overexpression will result in short kMTs with focused clusters of

kinetochores, one near to each SPB (Figure 1B, right). As shown

in Figure 1D, kinetochore clusters were indeed tightly focused

within each half-spindle and much closer to each SPB. Spindles

overexpressing Cin8p also have increased length due to in-

creased motor sliding between oppositely oriented central

spindle non-kMTs (also known as interpolar MTs) (Saunders

et al., 1997) (Figure S4). Despite the spindles being longer, kinet-

ochores in Cin8p overexpressing cells were still �50% closer to

SPBs than WT controls (Figure 1D), consistent with Cin8p over-

expression resulting in shorter kMTs (Figure S4). We conclude

that Cin8p and, to a lesser extent, Kip1p promote net kMT

plus-end disassembly as judged by kinetochore position.

GFP-Tubulin Fluorescence Confirms that kMTsAre Longer in cin8D MutantsIf Cin8p promotes net kMT plus-end disassembly, then CIN8 de-

letion will result in longer kMTs, producing a continuous ‘‘bar’’ of

fluorescent tubulin along the length of the spindle (Figure 2A,

right), rather than the WT fluorescent tubulin ‘‘tufts’’ that emanate

from each of the two SPBs (Figure 2A, left). In experiments with

GFP-Tub1, quantitative analysis of tubulin fluorescence in cin8D

mutants revealed a shift in fluorescence toward the spindle

equator, indicating that kMT length was increased (Figure 2A,

bottom). The distribution of GFP-Tub1 was quantitatively pre-

dicted in simulations using the same parameter set used to

model kinetochore organization in cin8D mutants (Figure 2A,

p = 0.22, Table S2). In addition, we found that the ratio of spindle

tubulin polymer signal to free tubulin signal outside of the spindle

area is 2.0:1 in WT spindles (n = 27) and 3.2:1 in cin8D mutant

spindles (n = 35), which represents an increase in tubulin polymer

relative to free tubulin of�62% in cin8D mutants as compared to

WT cells (p < 10�5). This suggests that the increased kMT length

in cin8D spindles is not the result of an overall increase of tubulin

level but rather reflects a thermodynamic shift toward increased

net kMT assembly.

Cryo-electron Tomography Confirms that kMTsAre Longer in cin8D MutantsTo directly visualize individual spindle MTs, we used cryo-elec-

tron tomography to reconstruct complete mitotic spindles from

WT and cin8D mutant spindles (Figure 2B; Movies S1 and S2).

To control for the moderate spindle length shortening in cin8D

mutants, we selected spindles of similar length in the WT and

mutant cell populations. Consistent with model predictions, we

found a substantial increase in mean MT length in cin8D mutant

spindles as compared to WT spindles (Figure 2B, 41% increase

in mean overall length, p = 0.0007, statistical consistency be-

tween cells confirmed by ANOVA in Table S1). Total non-kMT

number also increased in cin8D as compared to WT spindles

(42% increase in total MT number, p = 0.002), demonstrating

that the total polymer level in the cin8D cells is increased relative

to WT cells. Interestingly, the mean length of the eight longest

MTs in each spindle, presumably iMTs, is not statistically differ-

ent between WT and cin8D mutant cells (WT = 931 ± 81 nm

[mean ± SEM, n = 33 MTs]; cin8D = 1141 ± 25 nm [n = 41 MTs],

p = 0.05). This result suggests that deletion of CIN8 most signifi-

cantly affects the kMT length rather than iMT length.

In summary, the electron microscopy results independently

confirm the model predictions and the light microscopy studies

by demonstrating that kMTs are indeed longer in cin8D cells.

We conclude that Cin8p participates in a process that promotes

net kMT disassembly.

A Cin8p Motor-Domain Mutant Has Increased kMTLengthTo test whether kMT length regulation requires the Cin8p motor

domain, the organization of fluorescent tubulin was examined in

the cin8-F467A mutant (Gheber et al., 1999). Previous studies

found that the cin8-F467A mutation reduced the binding of

Cin8p to microtubules in vitro by 10-fold (Gheber et al., 1999).

Similar to the cin8D mutants, analysis of GFP-Tub1 distribution

in the cin8-F467A mutants produced a continuous bar of fluores-

cent tubulin along the length of the spindle (Figure 2C, right),

rather than the WT fluorescent tubulin tufts (Figure 2C, left).

The similarity between the cin8D and the cin8-F467A mutant

phenotypes suggests that the kMT disassembly promoting ac-

tivity of Cin8p requires motor binding to kMTs. Furthermore,

since our previous studies showed that net kMT assembly is pro-

moted when kMTs are short and suppressed when kMTs are

long (i.e., when kMT plus ends extend into the equatorial region,

Figure 1B), we also conclude that Cin8p-mediated suppression

of kMT assembly is specific to longer kMTs.

Cin8p Mediates the Gradient in Net kMT Assemblyas Measured by GFP-Tubulin FRAPTo further test whether Cin8p specifically suppresses assembly

of longer kMTs and thus mediates a gradient in net kMT assem-

bly, we measured the spatial gradient in tubulin turnover within

the mitotic spindle. In our previous work, we found that tubulin

turnover, as measured by spatially resolved GFP-tubulin fluores-

cence recovery after photobleaching (FRAP), is most rapid

where kMT plus ends are clustered in WT cells (Pearson et al.,

2006) . If Cin8p mediates a gradient in net kMT assembly, then

its deletion is predicted to result in loss of the gradient in FRAP

half-time. As shown in Figure 2D, deletion of CIN8 results in

loss of the tubulin turnover gradient, as predicted by the model

(Figure 1B, center). In general, the kMTs remain dynamic (overall

t1/2 = 46 ± 15 s integrated over the half-spindle in cin8D [n = 11],

compared to t1/2 = 63 ± 30 s for WT [Pearson et al., 2006], [n = 22;

p = 0.03; Figure S5]) and have a high fractional recovery (�90%

for cin8D, compared to�70% for WT [Maddox et al., 2000; Pear-

son et al., 2006]).

In simulating the cin8D GFP-tubulin FRAP experiment, the

best fit between theory and experiment is achieved with a flat-

tened spatial gradient in net kMT plus-end assembly (e.g., a flat-

tened catastrophe gradient) and with values for MT plus-end

growth and shortening rates that are slightly higher than in WT

simulations (Table S2). This result explains why FRAP is relatively

rapid along the entire length of the half-spindle in the cin8D mu-

tants and is consistent with recent studies that find increased

chromosome oscillation velocities upon deletion of the human

kinesin-8 motor Kif18A (Stumpff et al., 2008; reviewed in Gardner

et al., 2008). In summary, we find that the tubulin-FRAP studies

confirm that Cin8p mediates a spatial gradient in kMT assembly

dynamics.

Deletion of the Kinesin-8 Motor Kip3p Does Not PerturbCongressionRecent studies found that the kinesin-8 molecular motor Kip3p

acts as a length-dependent depolymerase in vitro (Varga et al.,

2006), and further studies have implicated the human kinesin-8

Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc. 897

Figure 2. Cin8p Promotes kMT Disassembly

(A)CIN8 deletion results inflattening of the GFP-tubulinfluorescencedistribution andshiftingof fluorescence toward the spindle equator, suggesting that kMT lengths

are increased in the mutant (red, Spc29-CFP SPB marker; green, GFP-Tub1 MT marker) (scale bar 1000 nm, error bars, SEM). All model parameters as in Table S2.

(B) Cryo-electron tomography reveals increased mean MT length and number in cin8D spindles (n = 5 spindles, mean spindle length = 1387 nm), relative to WT spin-

dles (n = 4 spindles, mean spindle length = 1265 nm) (error bars, SEM).

(C)Similar to the CIN8 deletionmutant, themotor-domainmutant, cin8-F467A, whichhas reduced affinity for MTs, results inflattening of the GFP-tubulinfluorescence

distribution and shifting of fluorescence toward the spindle equator (error bars, SEM).

(D)CIN8deletioneliminates thecharacteristicgradient inGFP-tubulinFRAPrecoveryhalf-time,consistentwithdisruptionof thegradient innetkMTassembly (errorbars,SEM).

(E) In contrast to the cin8D mutants, deletion of the kinesin-8 depolymerase KIP3 does not significantly perturb kinetochore microtubule (kMT) organization (error bars, SEM).

898 Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc.

molecular motor Kif18A in the regulation of chromosome oscilla-

tion amplitude (Stumpff et al., 2008) and chromosome congres-

sion (Mayr et al., 2007). Thus, we asked whether Kip3p deletion

would perturb kMT length organization in the yeast mitotic spin-

dle. Interestingly, deletion of KIP3 does not produce the contin-

uous bar of fluorescent tubulin along the length of the spindle

that is characteristic of cin8D spindles but rather shows WT fluo-

rescent tubulin tufts (Figure 2E). Although microtubule organiza-

tion appears WT, spindle lengths are moderately increased in

kip3D cells, as previously reported (Straight et al., 1998) (mean

spindle lengths in Figure 2E, WT: 1.60 ± 0.22; kip3D: 1.97 ±

0.15 mm). Thus, Kip3p regulates spindle length, possibly through

regulation of iMT lengths, while Cin8p regulates kMT length with-

out a measurable effect on iMT lengths. Regardless of effects on

iMTs, the effect of Kip3p on kMTs is relatively weak compared to

the effect of Cin8p and is insufficient to establish congression in

the absence of Cin8p.

Cin8p Promotes Shortening of Astral MTsin the CytoplasmMTs are densely packed in the yeast mitotic spindle (Figure 2B),

and so it is difficult to resolve individual spindle MTs via fluores-

cence microscopy. In contrast, yeast aMTs are normally much

fewer in number (1–3) and splayed apart (Figure 3A) (Gupta

et al., 2002; Shaw et al., 1997). Because Kip3p is known to affect

aMT dynamics (Cottingham and Hoyt, 1997; Gupta et al., 2006;

Miller et al., 1998), but the effect of Cin8p is not known, we then

examined the effect of motor deletion on aMTs outside of the

Figure 3. Cin8p Promotes Astral MT Disas-

sembly

(A) Astral MTs (aMTs) extend from the SPBs into

the cytoplasm and are fewer in number than spin-

dle microtubules.

(B) aMT lengths were measured via GFP-tubulin

fluorescence (white arrows point to plus ends;

red, Spc29-CFP pole marker; green, GFP-tubulin)

(scale bar 500 nm).

(C) aMT lengths are increased in cin8D cells as

compared to WT spindles. Cytoplasmic overex-

pression of Cin8p results in shorter aMTs, whether

overexpression is global, as in GAL1-CIN8 overex-

pression experiments, or if overexpression is local

in the cytoplasm, as in experiments with mutant

Cin8p lacking the nuclear localization signal

(cin8-nlsD). Both kip3D and cin8-F467A mutants

have increased aMT lengths relative to WT cells,

similar to cin8D cells (error bars, SEM).

mitotic spindle. Indeed, we found that

aMT lengths were statistically longer in

kip3D mutants as compared to WT cells

(Figures 3B and 3C), consistent with pre-

vious studies (Cottingham and Hoyt,

1997; Gupta et al., 2006).

A recent report suggests that Cin8p

plays a role in spindle positioning through

an aMT-dependent mechanism, and so,

coupled with our results for kMTs, we hy-

pothesized that Cin8p also suppresses aMT assembly (de Gram-

ont et al., 2007; Geiser et al., 1997). Using GFP-tubulin fluores-

cence microscopy, we measured the length of individual aMTs

in the cytoplasm (Figure 3B) (Carminati and Stearns, 1997; Gupta

et al., 2002; Shaw et al., 1997). Consistent with the behavior of

kMTs, aMTs were longer in cin8D mutants relative to WT cells

(Figures 3B, 3C and S6). In addition, a previous study showed

that aMT numbers are not decreased in cin8D mutants (de

Gramont et al., 2007), consistent with an overall increase in

both kMT and aMT polymer in cin8D mutants. Interestingly,

aMT lengths in the cin8-F467A mutants with reduced binding

of Cin8p to microtubules were also statistically longer than WT

aMTs (Figure 3C). Conversely, overexpression of Cin8p resulted

in shorter aMTs (Figures 3B, 3C, and S6).

Since the tubulin polymer level in the cin8D cells increases in

both the nucleus and the cytoplasm, these results argue against

a simple repartitioning of tubulin (or some other Cin8p-dependent

assembly-promoting factor) from the cytoplasm into the nucleus

in response to CIN8 deletion. Conversely, MT polymer levels de-

crease in both compartments upon Cin8p overexpression.

One possible reason why both kMTs and aMTs are longer in

cin8D cells is that CIN8 deletion indirectly promotes MT assem-

bly globally. To test this hypothesis, we shifted Cin8p from the

nucleus to the cytoplasm while keeping the overall Cin8p expres-

sion level approximately constant. This shift was achieved by de-

leting the nuclear localization sequence (NLS) of Cin8p (as previ-

ously described) (Hildebrandt and Hoyt, 2001). Because budding

yeast undergoes a closed mitosis, deleting the NLS decreases

Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc. 899

the nuclear Cin8p concentration and increases the cytoplasmic

Cin8p concentration (Hildebrandt and Hoyt, 2001). We found

that cin8-nlsD spindle MTs had a flat GFP-Tub1 fluorescence dis-

tribution, similar to cin8D cells (i.e., no tufts, Figure S7A). This re-

sult is consistent with net kMT assembly in the absence of Cin8p

locally in the nucleus (Figures 3B and S7A). Importantly, and in

contrast to cin8D cells, aMT lengths in cin8-nlsD cells were

shorter than in WT cells (Figures 3B and 3C, p < 0.001,

Figure S7C), consistent with elevation of Cin8p concentration

locally in the cytoplasm. These results indicate that Cin8p acts lo-

cally in a given cellular compartment, rather than globally through-

out the entire cell, to influence the local MT assembly state.

Together, these results suggest that Cin8p and Kip3p act sim-

ilarly to suppress assembly of aMTs outside of the mitotic spin-

dle. We then asked how Cin8p could act to promote kinetochore

congression through suppression of kMT plus-end assembly,

while Kip3p appears to regulate spindle length, possibly through

suppression of iMT plus-end assembly.

A Model for Cin8p Motor Interaction with kMTsBecause Cin8p requires microtubule binding to promote length-

dependent kMT plus-end disassembly, we hypothesized that

Cin8p acts directly on kMT plus ends, either by itself or with

a binding partner. To test the direct-interaction hypothesis, we

first predicted the spatial distribution of Cin8p motors on kMTs

via computational modeling. As a starting point, we extended

our previous model for individual kMT plus-end dynamics (Gard-

ner et al., 2005) to also include the dynamics of microtubule-

associated kinesin-5 molecular motors. This motor model as-

sumes that motors reversibly attach and detach, crosslink

MTs, and move toward MT plus ends (Movies S3 and S4; Figures

S8 and S9; Tables S3–S6) (Gheber et al., 1999; Kapitein et al.,

2005; Kashina et al., 1996; Valentine et al., 2006).

Cin8-GFP Is Distributed in a Gradient along kMTsAs shown in Figure 4A, this simple model for motor dynamics

predicts that, at steady-state, a substantial fraction of the motors

crosslink parallel kMTs emanating from the same SPB (green). In

the simulation, these motors walk to a kMT plus end and follow

the end as it grows until catastrophe occurs, at which point the

motor head detaches (Movie S4). By repetitive photobleaching

of Cin8-3XGFP-labeled spindles, we were able to experimentally

observe spindle-associated Cin8p motor dynamics in the

‘‘speckle’’ regime. Similar to the motor model simulation, we ob-

served motor movement in the spindle with a mean velocity of

58 ± 24 nm/s (mean ± standard deviation [SD], n = 10) (Figure 4B;

see Figure S8 and Supplemental Data).

To test the motor model, we predicted the distribution of Cin8-

GFP fluorescence in live cells as a function of spindle position

and then measured it experimentally. As shown in Figure 4C,

the motor model is able to quantitatively reproduce the experi-

mentally observed Cin8-GFP motor fluorescence distribution

(green) relative to kinetochores as measured by Ndc80-cherry

fluorescence (red) (p = 0.35). We found that in order to correctly

reproduce the Cin8-GFP fluorescence distribution, simulated

motors are required to track growing but not shortening kMT

plus ends, resulting in a slight, but detectable, shift in the peak

of motor-associated fluorescence away from kinetochores and

900 Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc.

toward SPBs (see Supplemental Data for analysis of various al-

ternative models, Figure S9). In the motor model, this shift is

the result of the increased motor off-rate imposed by shortening

kMT plus ends within the kinetochore clusters (Figure 4C, red). In

benomyl-treated spindles with stabilized microtubules, this shift

is decreased, such that the motor-associated fluorescence is

nearly coincident with kinetochores (Figure S10). Thus, the re-

sults with benomyl further support the hypothesis that kMT

plus-end assembly dynamics control motor dynamics, in partic-

ular motor detachment from kMT plus ends.

The Kip1-GFP fluorescence distribution was qualitatively sim-

ilar to the Cin8-GFP fluorescence distribution but was relatively

less focused into two clusters, consistent with a weaker affinity

of Kip1p for MTs (Figure S11).

By normalizing the Cin8-GFP fluorescence to the local kMT

density, we then calculated the Cin8-GFP density on a per

kMT basis (see Experimental Procedures for details). As shown

in Figure 4D, Cin8-GFP concentration per kMT gradually in-

creases with increasing distance from the SPB, as predicted

by the motor model. We conclude that ab-tubulins at the plus

ends of longer kMTs are more likely to be associated with

Cin8-GFP than ab-tubulins at the plus ends of shorter kMTs.

Cin8-GFP Turnover Is Most Rapid near kMT Plus EndsIf motors rapidly detach from shortening kMT plus ends, then the

rate of Cin8-GFP turnover on the spindle should be fastest in the

vicinity of shortening kMT plus ends. Specifically, the motor

model predicts there will be a spatial gradient in Cin8-GFP

FRAP half-time that is very similar to the GFP-tubulin FRAP

half-time gradient that we reported previously (Pearson et al.,

2006) (Figure 2C). Alternatively, the Cin8-GFP FRAP half-time

could be longest where kMT plus ends are clustered, indicative

of high-affinity binding to the kinetochore (Tytell and Sorger,

2006), which would also in principle explain the gradient in

Cin8-GFP intensity observed experimentally in Figure 4C. We

performed kinesin-5-GFP FRAP experiments and found that mo-

tor turnover is most rapid in the location of kMT plus-end cluster-

ing (Figures 4E and S12; p < 1 3 10�10 as compared to half-times

near the SPBs), suggesting that Cin8p motors rapidly detach

from shortening kMT plus ends. Mean Kip1-GFP FRAP half-

times were �50% faster than Cin8-GFP recovery half-times

(Figure S12), again suggesting that Kip1p has a lower affinity

for microtubules than Cin8p. The Cin8-GFP FRAP results are

all consistent with a model in which motors frequently interact

with kMT plus ends in a length-dependent manner.

To summarize our studies of spindle-bound kinesin-5

dynamics, we find that kMT-bound kinesin-5 motors are dis-

tributed in a spatial gradient along kMTs, with their concentra-

tion increasing with increasing distance from the SPB. Consis-

tent with the observed gradient, we also find that kinesin-5

motors frequently interact with, and are controlled in their de-

tachment from the MT lattice by, dynamic kMT plus ends. Be-

cause Cin8p suppresses assembly of longer kMTs to mediate

kinetochore congression, while Kip3p appears to act primarily

to regulate metaphase spindle length, we then asked how

the behavior of the kinesin-5 motor Cin8p compares to the

behavior of the kinesin-8 motor Kip3p in the mitotic spindle

environment.

Figure 4. Cin8p Accumulates on kMTs in a Length-Dependent Manner and Frequently Interacts with kMT Plus Ends

(A) In simulation, kinesin-5 motors crosslink both antiparallel-oriented microtubules (left, magenta) and parallel-oriented microtubules (right, green). Simulated

motors crosslinking parallel-oriented microtubules move to and frequently interact with kMT plus ends.

(B) Cin8-3XGFP motor movement can be observed in the spindle (horizontal scale bar, 1000 nm; vertical scale bar, 20 s). The three arrows (yellow, cyan, and

magenta) indicate three time points in the movement of a Cin8-3XGFP fluorescent spot that moves in the plus-end direction.

(C) Cin8p motors concentrate near kinetochores both experimentally and in simulation (red, Ndc80-cherry kinetochore marker; green, Cin8-GFP) (scale bar

500 nm, error bars, SEM).

(D) Experimentally and in simulation, Cin8-GFP fluorescence normalized to the number of tubulin polymer-binding sites increases for longer kMTs (error bars,

SEM).

(E) Cin8-GFP FRAP half-time gradient (error bars, SEM).

Cin8-GFP Concentrates near kMT Plus Ends, whileKip3-GFP Distribution Is DiffuseAs shown in Figure 5A and described above, Cin8-GFP concen-

trates near kMT plus ends. This localization is reproduced in sim-

ulation because a substantial fraction of the homotetrameric

Cin8 motors crosslink parallel kMTs emanating from the same

SPB and then walk efficiently toward the plus ends of both cross-

linked microtubules. In contrast, motors crosslinking antiparallel

microtubules in the simulation (i.e., microtubules attached to op-

posite SPBs, Figure 4A, magenta) are not able to make progress

Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc. 901

toward the plus ends of either microtubule to which they are at-

tached, as the motor heads walk in opposite directions. These

antiparallel attached motors quickly stall and remain stationary

until the detachment of one or both heads. Interestingly, since

iMTs run nearly the full length of the spindle, their plus ends re-

side in a highly antiparallel environment, with �4 iMT plus ends

surrounded by �20 antiparallel MTs attached to the opposite

SPB. Thus, the processivity of the tetrameric kinesin-5 motor is

naturally limited near to iMT plus ends due to the bipolar cross-

linking properties of the motor.

In contrast, dimeric Kip3p motors are not bipolar, and thus

their processivity is not hindered by crosslinking to antiparallel

microtubules. In this case, the longer iMTs act as larger anten-

nas, attracting larger numbers of plus-end-directed Kip3p mo-

tors to their ends (Varga et al., 2006). As shown in Figure 5A,

while Cin8-GFP is specifically concentrated near to kMT plus

ends, Kip3-GFP is more diffusely distributed along the length

Figure 5. Relative Distribution of Cin8-GFP and Kip3-GFP(A) The experimental and simulated distribution of Cin8-GFP and Kip3-GFP

(error bars, SEM).

(B) In simulations, the crosslinking properties of Cin8p frustrates its processiv-

ity toward the plus ends of interpolar (iMT) plus ends, such that Cin8p visits to

iMT plus nds are rare relative to kMT plus-end visits.

902 Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc.

of the spindle. Importantly, the relative concentration of Kip3-

GFP is higher near to where the iMT plus ends are located

(Figure 5A) as compared to Cin8-GFP, whose relative concentra-

tion is higher near to where kMT plus ends are located

(Figure 5A).

To test this interpretation of the localization data, simulations

were run that reproduced both Cin8-GFP and Kip3-GFP localiza-

tion in the spindle (Figure 5A). In these simulations, Cin8-GFP is

a bipolar MT crosslinking motor with simulation parameters as

described above, and Kip3p is a slightly faster monopolar non-

crosslinking motor (Kip3p motor velocity = 75 nm/s; Gupta et al.,

2006) with otherwise identical simulation parameters. In these

simulations, �90% of Cin8p motor visits to all MT plus ends

were to kMT plus ends specifically, while the majority of Kip3p

plus-end visits were to iMT plus ends (�60%, Figure 5B). Thus,

Cin8p molecular motors mediate congression by suppressing

the assembly of kMT plus ends, while Kip3p appears to act pri-

marily to limit assembly of iMT plus ends. The two different

outcomes can be modeled simply by either including bipolar

crosslinking (Cin8p) or not (Kip3p).

Cin8-GFP Motors on aMTsBecause Cin8p promotes aMT disassembly (Figure 3), we con-

sidered whether the motor model applies to cytoplasmic aMTs

as well. In contrast to the densely packed kMTs, 1–3 individual

aMTs can be readily observed in the cytoplasm so that individual

motor interaction with aMTs should also be readily observable.

Using timelapse microscopy we were able to visualize Cin8-

NLSD-3XGFP interacting with 1–3 individual aMTs. As shown

in Figure 6A (left), Cin8-NLSD-3XGFP moves persistently in the

plus-end direction along stationary mcherry-Tub1-labeled

aMTs (Movie S6). Motors did not move in the minus-end direc-

tion, consistent with the motor model and the inferred motor be-

havior on kMTs. The mean motor velocity was 15 ± 3.3 nm/s

(mean ± SD, n = 8), which is similar to previously reported aMT

plus-end growth rates of 8 ± 23 nm/s (Carminati and Stearns,

1997; Gupta et al., 2002, 2006; Huang and Huffaker, 2006;

Shaw et al., 1997), suggesting that motors track growing aMT

plus ends. Since Cin8-GFP was distributed along kMTs in a gra-

dient of increasing motor with increasing distance from the SPB,

we were interested to see whether a similar motor gradient oc-

curs on aMTs. As expected from the motor model and similar

to the behavior of motors on kMTs, we found that Cin8-NLSD-

GFP fluorescence is distributed along the length of aMTs with

a peak fluorescence shifted slightly away from aMT plus ends

(Figures 6B and 6C). We then normalized the motor fluorescence

to the aMT density and, as shown in Figure 6D, found a spatial

gradient of Cin8-NLSD-GFP that is very similar to the gradient

observed on kMTs and to the gradient predicted by the motor

model. An interesting feature of both the model and the experi-

ment is a slight dip in the motor concentration at the peak aMT

plus-end location (gray arrow in Figure 5D). This dip was also ap-

parent in the kMT data and simulation and again suggests that

dynamic MT plus ends mediate motor detachment from the

MT lattice. We conclude that Cin8p interacts frequently with

aMT plus ends in a manner similar to its interaction with kMT

plus ends and consistent with the motor model. We then asked

if the spatially regulated interaction of Cin8p with kMT plus

Figure 6. Cin8p Walks Processively toward MT Plus Ends, and Its

Distribution on aMTs Mirrors Its Distribution on kMTs

(A) Cin8-NLSD-3XGFP (left, green) moves in the plus-end direction on aMTs

and frequently interacts with aMT plus ends. The three arrows (yellow, cyan,

and magenta) indicate three time points in the movement of a Cin8-GFP fluo-

rescent spot that moves in the plus-end direction (red, tubulin-cherry) (horizon-

tal scale bar, 1500 nm; vertical scale bar, 50 s).

(B) Cin8-NLSD-3XGFP (green) on aMTs (tubulin-cherry, red).

(C) Cin8-NLSD-GFP concentrates near the plus ends of longer aMTs (error

bars, SEM).

(D)Experimentallyand insimulation,Cin8-GFPfluorescencenormalizedtothenum-

ber of tubulin polymer-binding sites increases for longer aMTs (error bars, SEM).

ends, together with the disassembly-promoting activities of

Cin8p, could act to mediate chromosome congression in the

yeast metaphase spindle.

Cin8p-Mediated ‘‘Self-Organized’’ Model for MetaphaseKinetochore OrganizationThe model for kinesin-5 motor dynamics that best agrees with

the experimental data described above is one in which motors

bind randomly to kMTs (Figure 7A, top) and then walk toward

kMT plus ends, where they act to promote net kMT disassembly

(Figure 7A, middle). The longer the MT, the more sites there are

for motors to attach, which results in more motors at the plus

end, and the more that assembly will be disrupted. Once net dis-

assembly occurs (e.g., via a catastrophe), then motors detach

from the shortening kMT plus end (Figure 7A, bottom). Impor-

tantly, because simulated motors bind randomly to microtubules

and are plus end directed, the motor model predicts that the

number of motor interactions at kMT plus ends will increase

with increasing kMT length, as shown recently for kinesin-8

molecular motors in vitro (Mayr et al., 2007; Varga et al., 2006)

(Figures 4D and 6D).

To determine if simulated Cin8p motors could mediate chro-

mosome congression via their disassembly-promoting activity

at kMT plus ends, we allowed the Cin8p motors to promote ca-

tastrophe as they concentrate on kMT plus ends in the motor

model simulation as described above. In this self-organized

model, there is no externally imposed ‘‘catastrophe gradient’’

to regulate kMT lengths (as in Figure 1B), but rather the catastro-

phe-promoting motors naturally concentrate on kMT plus ends

in a length-dependent manner. Thus, by starting the simulation

with a random organization of motors and kMT lengths (Fig-

ure 7B, top), we found that self-organization of kinetochores

into a bilobed metaphase configuration arises naturally when

Cin8p motors promote catastrophe at kMT plus ends

(Figure 7B, bottom; Movie S5). Here, the catastrophe-promoting

Cin8p motors self-organize a simulated bilobed spindle in which

both Cin8-GFP and Ndc80-cherry fluorescence distributions are

qualitatively consistent with experimental results (Figure 7C). By

allowing motor presence at the kinetochore to directly promote

catastrophe, the theoretical kMT plus-end catastrophe gradient

model (Figure 1B) is closely reproduced (Figure 7D). As in

Figure 1B, this gradient serves to establish the bilobed distribu-

tion of kinetochores into the two distinct clusters that are charac-

teristic of the congressed metaphase spindle (Figure 1A). Similar

to experimental results in cinD mutants, reducing the motor

number in the self-organized simulation disrupts the gradient in

kMT assembly and thus leads to disorganization of the spindle

(Figure S13).

DISCUSSION

Our results demonstrate that kinesin-5 motors, in particular

Cin8p, promote net kMT and aMT disassembly in vivo. Since

net kMT assembly is specifically suppressed for longer kMTs

(i.e., whose plus ends are near the equator) during yeast meta-

phase, kinesin-5 motors must be mediating their effect, either

alone or with binding partners, most strongly on longer kMTs.

It is this length-dependent regulation of net kMT plus-end

Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc. 903

Figure 7. A Self-Organized Model for Cin8p Motor-Mediated Spindle Organization

(A) A model for interaction of Cin8p motors with kMT plus ends: Cin8p motors that crosslink parallel microtubules are plus end directed (top). Cin8p concentrates

on longer kMT plus ends to directly promote kMT disassembly (middle). kMT depolymerization then promotes motor detachment (bottom).

(B) Starting with a random distribution of kinetochores and motors in the spindle (top), motor-mediated promotion of kMT plus-end disassembly can organize

the spindle into a typical metaphase bilobed kinetochore configuration as motors concentrate in a length-dependent fashion onto kMT plus ends (bottom and

Movie S5).

(C) Simulated images of Cin8-GFP (green) and Ndc80-cherry (red). At simulation start (t = 0), motor and kinetochore fluorescence is randomly distributed in the

spindle (left). After t = 135 s, simulated fluorescence distributions qualitatively reproduce experimental results (top right, simulated image; bottom, quantitative

comparison to experimental data). Model parameters are in Table S7. (Error bars, SEM.)

(D) The self-organized model results in a gradient of catastrophe frequency (red triangles) that is similar to the theoretical catastrophe gradient depicted in

Figure 1B (red line).

assembly that establishes the congressed state of chromo-

somes that is characteristic of metaphase. These results are sur-

prising to us since the only established activity of kinesin-5 is its

well-known antiparallel MT sliding activity, and, to our knowl-

edge, no effect on MT assembly has been reported previously.

We also found that Cin8-GFP interacts frequently with MT plus

ends in vivo and exists in a spatial gradient on MTs. The close

correspondence of the gradient in net kMT assembly and the

gradient in motor distribution strongly suggests that Cin8p,

either alone or with a binding partner, directly promotes kMT

disassembly via its presence at the kMT plus end. In addition,

this conclusion is strengthened by the close similarity between

the cin8D mutant and the cin8 motor-domain mutant (F467A)

phenotypes.

It is interesting to consider the consequences of the disassem-

bly promotion for spindle length regulation. It is well established

that kinesin-5 promotes spindle pole separation by generating

an outward extensional force, presumably via crosslinking of

antiparallel MTs. The newly identified disassembly-promoting

activity shortens kMTs and thereby should generate an inward

904 Cell 135, 894–906, November 28, 2008 ª2008 Elsevier Inc.

pulling force on the spindle poles via stretching of the intervening

chromatin between the sister kMT plus ends. Thus, the two ac-

tivities of Cin8p antagonize each other and are expected to result

in stable spindle pole separation during yeast metaphase.

As described above, the model for kinesin-5 motor dynamics

in the metaphase budding yeast mitotic spindle that best agrees

with experimental data is one in which motors bind randomly to

kMTs (Figure 7A, top) and then walk toward kMT plus ends,

where they act to promote net kMT disassembly (Figure 7A, mid-

dle). Here, because longer kMTs have a larger number of possi-

ble motor-binding sites, the motor model predicts that the num-

ber of motor interactions at kMT plus ends will increase with

increasing kMT length, as shown recently for kinesin-8 molecular

motors in vitro (Mayr et al., 2007; Varga et al., 2006). Interest-

ingly, the plus ends of iMTs, which are the longest MTs in the

yeast mitotic spindle, may attract fewer Cin8p motors than the

plus ends of kMTs simply because iMT plus ends are surrounded

by potential antiparallel motor attachments, frustrating the plus-

end motility of bipolar crosslinking Cin8p motors. This explana-

tion is supported by results for the monopolar depolymerizing

motor Kip3p, whose localization and deletion phenotypes are

distinct from those of Cin8p. We found that Kip3-GFP behavior

could be reproduced by taking the Cin8-GFP simulation and

turning ‘‘off’’ the crosslinking. In this case, the noncrosslinking

motor tends to accumulate more onto iMTs and less so onto

kMTs. The simple physical feature of bipolar MT crosslinking,

enabled in the tetrameric Cin8p and disabled in the dimeric

Kip3p, is sufficient to explain all the experimental results.

The simplest molecular mechanism for length-dependent MT

disassembly is that the kinesin-5 motor itself acts directly to pro-

mote MT plus-end disassembly. We speculate that mechanical

stress between walking motor head domains would stress

tubulin-tubulin bonds to destabilize the lattice and promote MT

disassembly. Alternatively, kinesin-5 could carry a disassem-

bly-promoting binding partner to promote net disassembly at

MT plus ends, although to our knowledge there are no known

cargoes that are transported by kinesin-5 motors.

Either way, the role of kinesin-5 motors in regulating kMT as-

sembly dynamics is a new property that we have now identified

for a motor previously known only as a sliding motor that acts be-

tween antiparallel MTs. Because of the potent effect that CIN8

deletion has on kinetochore organization, it seems unlikely that

a significant Cin8p-independent pathway will be found to also

promote length-dependent disassembly. The effects of kine-

sin-5 on MT assembly will be important to consider as anticancer

drugs directed toward inhibiting kinesin-5 sliding activity are

presently in clinical trials (Sudakin and Yen, 2007).

EXPERIMENTAL PROCEDURES

Yeast Strains and Cell Culture

All relevant genotypic information can be found in Table S8. Genes of fusion

proteins remained under control of their endogenous promoters. Cell growth

techniques and conditions were performed as previously described (Bouck

and Bloom, 2007; Gardner et al., 2005; Pearson et al., 2003, 2004). The

CIN8 NLS deletion was performed as previously described (Hildebrandt and

Hoyt, 2001). Overexpression of Cin8p from the PGAL1 promoter was performed

as previously described (Saunders et al., 1997). The CIN8-3XGFP was made

by PCR amplification of 3XGFP from a plasmid, and then the linear PCR prod-

uct was integrated at the endogenous CIN8 gene. The cin8-F467A strain was

made and verified as previously described (Gheber et al., 1999). Benomyl

treatment for stabilization of kMT plus-end dynamics was performed as previ-

ously described (Pearson et al., 2003).

Fluorescence Imaging and Photobleaching

Fluorescence imaging and photobleaching experiments were performed as

previously described (Pearson et al., 2001, 2006). Astral MT lengths were as-

sessed by measuring the lengths of GFP-Tub1- or mcherry-Tub1-labeled

aMTs in which both the plus and minus ends were clearly visible within one fo-

cal plane. Counting of Cin8p and Kip1p on the mitotic spindle was completed

as previously described (Joglekar et al., 2006).

Image Analysis

Average fluorescence distributions calculated over normalized spindle lengths

were obtained as previously described (Gardner et al., 2005).

In FRAP experiments, FRAP half-times were resolved according to spindle

position, as previously described (Maddox et al., 2000; Pearson et al., 2006).

Reported half-spindle FRAP half-times were calculated by averaging over all

spindle positions in the photobleached half-spindle.

Cin8-GFP fluorescence normalized to the number of tubulin-binding sites

(Figures 4D and 6D) is calculated as follows. For Figure 4D, the tubulin decay

function was calculated by assuming a tubulin-binding site fraction of 1.3 at the

SPBs, which decays inversely with increasing Ndc80-cherry fluorescence

such that there remains a fraction of 0.3 binding sites at the spindle equator

(representing interpolar MTs). Then, Cin8-GFP fluorescence was normalized

to the number of tubulin-binding sites by dividing Cin8-GFP signal minus back-

ground by the tubulin decay function at each spindle position. A similar method

was used for Figure 6D, except that the tubulin decay function was calculated

directly from the distribution of aMT plus ends as is shown in Figure 6C (red). All

p values for relative comparisons of experimental data sets were calculated

using Student’s t test, unless otherwise noted. Quantitative comparisons of

simulations to experimental results were completed as previously described

(Sprague et al., 2003).

Simulation Methods

All simulations were run using MATLAB R7.1 (Natick, MA, USA). Detailed sim-

ulation methods are provided in the Supplemental Data.

Tomography Methods

Cells were prepared for electron microscopy using high-pressure freezing fol-

lowed by freeze substitution as previously described (Winey et al., 1995). Dual

axis electron tomography was carried out as described previously (O’Toole

et al., 2002).

In total, we recorded four WT spindles and five cin8D spindles. Individual mi-

crotubules and the position of the SPB central plaque were modeled from the

tomographic volumes. A projection of the three-dimensional (3D) model was

then displayed and rotated to study its 3D geometry. Microtubule lengths

were extracted from the model contour data using the program IMODINFO.

SUPPLEMENTAL DATA

Supplemental Data include Supplemental Results, thirteen figures, eight ta-

bles, and six movies and can be found with this article online at http://www.

cell.com/supplemental/S0092-8674(08)01239-7.

ACKNOWLEDGMENTS

The authors thank Dominique Seetapun for providing matlab kymograph code,

Drs. Tom Hays and Leah Gheber for helpful discussions, Dr. Jeff Molk for com-

ments on the manuscript, and Marybeth Anderson for assistance with bim1D

images. Plasmids and strains were kindly provided by Drs. S. Reed, B. Errede,

M.A. Hoyt, L. Gheber, and D. Pellman. This work was supported by NIH grant

GM071522 to D.J.O. K.B. is supported by NIH grant GM32238 and M.K.G. is

supported by NIH NRSA grant EB005568. E.T.O. is supported in part by grant

RR-00592 from the National Center for Research Resources of the NIH to

A. Hoenger. L.V.P. is supported by a SPIRE fellowship (GM00678).

Received: January 24, 2008

Revised: June 29, 2008

Accepted: September 23, 2008

Published: November 26, 2008

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